New Strategies and Screening Methods for Metalloproteinase Inhibition

描述（由申请人提供）：本提案的主要目标是：a)制备各种锌（II）金属蛋白的双齿磺胺抑制剂，b)开发一种基于细胞的筛选方法，以同时检测这些抑制剂对几种金属酶的影响。基质金属蛋白酶（MMPs）在炎症性疾病、中风、关节炎、癌症和心脏病的进展中发挥核心作用。所有MMPs都利用锌（II）离子催化水解结缔组织和其他生物基质。本项目的总体目标是开发改进的基质金属蛋白酶抑制剂（MMPi），利用锌（II）特异性磺胺螯合剂作为新的锌结合基团（ZBGs）。螯合磺胺将增强与锌（II）金属蛋白活性位点的结合，同时保留其他金属酶（如铁依赖性酶）。为了验证这一假设，这些抑制剂对MMPs的选择性将使用一种新的基于巨噬细胞的筛选方法进行评估。本提案的目标将通过以下具体目标来实现：1。简单螯合磺胺ZBGs的合成与表征。与复制MMP活性位点的生物无机模型配合物结合，磺胺zbg将提供这些配体对锌（II）离子的结合模式和亲和力的重要信息。2. 新型MMPi的设计与合成。利用我们实验室的生物无机-计算相结合的方法，基于新的磺胺ZBGs，将设计出有效和选择性的MMPi。3. 用荧光和比色法评价新型MMPi。新的MMPi将与多种重组MMPs进行比较。4. MMPi对巨噬细胞炎症模型中几种金属酶的筛选。MMPi将通过一种新的基于巨噬细胞的检测来检测，该检测将确定MMPs抑制剂对其他锌（II）依赖性金属酶、血红素-铁金属酶和非血红素-铁金属酶的选择性。通过使用这种方法并实现这些目标，我们将鉴定出比现有MMPi具有更大亲和力、选择性和生物相容性的新MMPi，从而鉴定出将在临床上成功的抑制剂。该项目的成功将产生至少两项极具创新性和高影响力的发现：a)首次使用锌（II）离子分子传感器中经常发现的锌（II）选择性螯合磺胺，将用于锌（II）依赖性金属蛋白抑制，b)开发基于细胞的金属蛋白抑制剂筛选方法，以评估化合物对生物环境中广泛的金属依赖性酶的影响。能够选择性抑制金属依赖性酶的药物的开发对于中风、关节炎、癌症和心脏病等疾病的治疗至关重要。该项目旨在发现选择性金属蛋白抑制剂，并开发一种简便的方法，通过这种方法可以快速评估这些抑制剂的选择性。公共卫生相关性：能够选择性抑制金属依赖性酶的药物的开发对中风、关节炎、癌症和心脏病等疾病的治疗至关重要。该项目旨在发现选择性金属蛋白抑制剂，并开发一种简便的方法，通过这种方法可以快速评估这些抑制剂的选择性。

DESCRIPTION (provided by applicant): The major objectives of this proposal are: a) to prepare a variety of bidentate sulfonamide inhibitors of zinc(II) metalloproteins, and b) develop a cell-based screening method for examining the effect of these inhibitors on several metalloenzymes simultaneously. Matrix metalloproteinases (MMPs) play a central role in the progression of inflammatory disease, stroke, arthritis, cancer, and heart disease. All MMPs utilize a zinc(II) ion to perform the catalytic hydrolysis of connective tissue and other biological substrates. The overall aim of this project is to develop improved matrix metalloproteinase inhibitors (MMPi) that utilize zinc(II)-specific sulfonamide chelators as novel zinc-binding groups (ZBGs). Chelating sulfonamides will enhance binding to the zinc(II) metalloprotein active site, while sparing other metalloenzymes (e.g. iron- dependent enzymes). To validate this hypothesis, the selectivity of these inhibitors for MMPs will be assessed using a novel macrophage-based screening methodology. The goals of this proposal will be achieved via the following specific aims: 1. The synthesis and characterization of simple, chelating sulfonamide ZBGs. Combined with bioinorganic model complexes that reproduce the MMP active site, sulfonamide ZBGs will provide essential information on the binding mode and affinity of these ligands for the zinc(II) ion. 2. The design and synthesis of novel MMPi. Using a combined bioinorganic-computational approach from our laboratory, potent and selective MMPi will be devised based on new sulfonamide ZBGs. 3. The evaluation of novel MMPi using fluorescence and colorimetric assays. New MMPi will be evaluated against a variety of recombinant MMPs. 4. The screening of MMPi against several metalloenzymes in a macrophage cell model of inflammation. MMPi will be examined in a novel macrophage cell-based assay that will determine the selectivity of the inhibitors for MMPs versus other zinc(II)-dependent metalloenzymes, heme- iron metalloenzymes, and non-heme-iron metalloenzymes. By using this approach and achieving these aims, we will identify new MMPi with greater affinity, selectivity, and biocompatibility than current MMPi, and thereby identify inhibitors that will be clinically successful. The success of this project will generate at least two highly innovative and high-impact findings: a) the first time that the use of zinc(II)-selective chelating sulfonamides, which are frequently found in zinc(II) ion molecular sensors, will be utilized for zinc(II)-dependent metalloprotein inhibition, and b) the development of a cell-based screening method for metalloprotein inhibitors that evaluates the effect of a compound against a broad set of metal-dependent enzymes in a biological setting. The development of drugs that can selectively inhibit metal-dependent enzymes is critical to the treatment of illnesses such as stroke, arthritis, cancer, and heart disease. This project seeks to discover selective metalloprotein inhibitors and develop a facile method by which the selectivity of these inhibitors can be rapidly assessed. PUBLIC HEALTH RELEVANCE: The development of drugs that can selectively inhibit metal-dependent enzymes is critical to the treatment of illnesses such as stroke, arthritis, cancer, and heart disease. This project seeks to discover selective metalloprotein inhibitors and develop a facile method by which the selectivity of these inhibitors can be rapidly assessed.